Cryptosporidium molnari n. sp. (Apicomplexa: Cryptosporidiidae) infecting two marine fish species,Sparus aurata L. and Dicentrarchus labrax L

Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.     

Goussia girellae n. sp. (Apicomplexa: Eimeriorina) in the opaleye, Girella nigricans

Goussia girellae n. sp. is described from the opaleye fish, Girella nigricans. Merogonic stages were observed in the apices of intestinal epithelial cells, in the lamina propria, and in extra-intestinal sites including liver, gills, and spleen. Gamonts were observed in the intestinal epithelial cells. Only unsporulated oocysts were detected in the intestine, and sporulation occurred when feces containing oocysts were incubated for 48 h in seawater at 21 degrees C. Oocysts are elongated (24.8 x 14.7 micron) with a wall about 200 nm thick and have no residuum, micropyle, or polar granule. Sporocysts are ellipsoid (8.5 x 4.5 micron), have a thin two-layered wall approximately 30 nm thick, and consist of two valves joined by a suture. Although moribund opaleye were also infected with Gyrodactylus sp., Cryptobia sp., Cardicola sp., and epitheliocystis organisms (chlamydia), all fish were heavily infected with G. girellae and morbidity was thus attributed to the coccidium.     

A redescription of Cryptosporidium galli Pavlasek, 1999 (Apicomplexa: Cryptosporidiidae) from birds

Cryptosporidium galli Pavlasek, 1999, described from the feces of birds, is redescribed with additional molecular and biological data. Oocysts are ellipsoidal, are passed fully sporulated, lack sporocysts, and measure 8.25 x 6.3 microm (range 8.0-8.5 x 6.2-6.4 microm) with a length-width ratio of 1.30 (n = 50). Oocysts are structurally similar to those of Cryptosporidium baileyi described from chickens, but in addition to being considerably larger than oocysts of C. baileyi, these oocysts infect the proventriculus in a variety of birds and not the respiratory tract. Oocysts were successfully transmitted from chickens to chickens, and morphologically similar oocysts also were observed in a variety of exotic and wild birds (Order Passeriformes, Phasianidae, Fringillidae, and Icteridae). Molecular and phylogenetic analyses at the 18S rRNA, HSP70, and actin gene loci demonstrate that this species is genetically distinct from all known species and genotypes of Cryptosporidium and, thus, was named C. galli.     

Cryptosporidium fayeri n. sp. (Apicomplexa: Cryptosporidiidae) from the Red Kangaroo (Macropus rufus)

The morphology and infectivity of the oocysts of a new species of Cryptosporidium from the faeces of the red kangaroo (Macropus rufus) are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts, and measure 4.5-5.1 microm (mean=4.9) x 3.8-5.0 microm (mean=4.3 microm) with a length to width ratio 1.02:1.18 (mean 1.14) (n=50). Oocysts were not infectious for neonate ARC Swiss mice. Multi-locus analysis of numerous unlinked loci demonstrated this species to be distinct (90.64%-97.88% similarity) from C. parvum. Based on biological and molecular data, this Cryptosporidium infecting marsupials is proposed to be a new species Cryptosporidium fayeri n. sp.     

The life cycle of Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) infecting chickens

The life cycle and morphology of a previously undescribed species of Cryptosporidium isolated from commercial broiler chickens is described. The prepatent period for Cryptosporidium baileyi n. sp. was three days post oral inoculation (PI) of oocysts, and the patent period was days 4-24 PI for chickens inoculated at two days of age and days 4-14 for chickens inoculated at one and six months of age. During the first three days PI, most developmental stages of C. baileyi were found in the microvillous region of enterocytes of the ileum and large intestine. By day 4 PI, most parasites occurred in enterocytes of the cloaca and bursa of Fabricius (BF). Mature Type I meronts with eight merozoites first appeared 12 h PI and measured 5.0 x 4.9 micrometers. Mature Type II meronts with four merozoites and a large granular residuum first appeared 48 h PI and measured 5.1 x 5.1 micrometers. Type III meronts with eight short merozoites and a large homogeneous residuum first appeared 72 h PI and measured 5.2 x 5.1 micrometers. Microgamonts (4.0 x 4.0 micrometers) produced approximately 16 microgametes that penetrated into macrogametes (4.7 x 4.7 micrometers). Macrogametes gave rise to two types of oocysts that sporulated within the host cells. Most were thick-walled oocysts (6.3 x 5.2 micrometers), the resistant forms that passed unaltered in the feces. Some were thin-walled oocysts whose wall (membrane) readily ruptured upon release from the host cell. Sporozoites from thin-walled oocysts were observed penetrating enterocytes in mucosal smears. The presence of thin-walled, autoinfective oocysts and the recycling of Type I meronts may explain why chickens develop heavy intestinal infections lasting up to 21 days. Oocysts of C. baileyi were inoculated orally into several animals to determine its host specificity. Cryptosporidium baileyi did not produce infections in suckling mice and goats or in two-day-old or two-week-old quail. One of six 10-day-old turkeys had small numbers of asexual stages only in the BF. Four of six one-day-old turkeys developed mild infections only in the BF, and sexual stages of the parasite were observed in only one of the four. All seven one-day-old ducks and seven two-day-old geese developed heavy infections only in the BF with all known developmental stages present.     

Chemical and physical factors affecting the excystation of Cryptosporidium parvum oocysts.

Cryptosporidium parvum oocysts were examined to ascertain excystation requirements and the effects of gamma irradiation. Oocysts and excysted sporozoites were examined for dye permeability and infectivity. Maximum excystation occurred when oocysts were pretreated with acid and incubated with bile salts, and potassium or sodium bicarbonate. Pretreatment with Hanks' balanced salt solution or NaCl lowered excystation; however, this effect was overcome with acid. Sodium ions were replaceable with potassium ions, and sodium bicarbonate was replaceable with sodium phosphate. Oocysts that received 200 krad irradiation excysted at the same rates as nonirradiated oocysts (95%), the excystation rates were lowered (50%) by 2,000 krad, and no excystation was observed by 5,000 krad. No differences were observed between the propidium iodide (PI) permeability of untreated oocysts and oocysts treated with 200 krad, while 92% of oocysts were PI positive after 2,000 krad. Most of the sporozoites exposed to 2,000 krad were not viable as indicated by the dye permeability assay. The oocysts irradiated with 200 and 2,000 krad infected cells, but no replication was observed. The results suggest that gamma-irradiated oocysts may still be capable of excystation and apparent infection; however, because the sporozoites could not reproduce they must not have been viable.     

Isolation and identification of Cryptosporidium from various animals in Korea. II. Identification of Cryptosporidium muris from mice

Each of SPF mice(Scl: ICR strain, 3-week-old males) was inoculated with 5 x 10(4) oocysts of Cryptosporidium by stomach tube. The oocysts were large type one which was previously isolated from Korean mice, and passaged in 3-week-old SPF mice. The patterns of oocyst discharge were monitored daily, and in order to observe the ultrastructure of developmental stages the stomach of the mice was examined by transmission electron microscopy (TEM) at 4 weeks post-inoculation. The prepatent period for 6 mice was 5.6 days post-inoculation on the average, and the patent period was 63.2 days. The number of oocysts discharged per day from the mice reached peak on day 36.6 post-inoculation on the average. A large number of oocysts were found in fecal samples obtained from inoculated mice on days 30-50 post-inoculation. C. muris was larger than C. parvum at almost every developmental stages, the size difference being 1.4 times in oocysts, 2.4 times in sporozoites, 1.6 times in merozoites, and 1.5 times in microgametes. The ultrastructural features of the attachment site of C. muris to the mucus cells were remarkably different from those of C. parvum and its closely related species. The anterior projection of the protozoa (C. muris), the outer aspect of which was surrounded by a thick filamentous process of the host cell, has not been reported at any developmental stages of C. parvum or its closely related species. The size of the oocysts of strain RN 66 was larger than that of Korean mice origin. The above results reveal that the large type Cryptosporidium of Korean mice origin is identified as Cryptosporidium muris and this type was named as C. muris (strain MCR).     

The ultrastructure of gametogenesis of cryptosporidium baileyi (eimeriorina; cryptosporidiidae) in the respiratory tract of broiler chickens (Gallus domesticus).

The ultrastructural features of sexual development of Cryptosporidium baileyi in the respiratory tract of experimentally infected broiler chickens were studied using transmission electron microscopy. Sexual stages of C. baileyi were seen attached to the tracheal epithelium and free in the tracheal lumen. These stages included intracellular type III merozoite-like stages, microgamonts, microgametes, macrogamonts, thin-walled oocysts, and thick-walled oocysts. These stages were developmentally similar to those observed for other Cryptosporidium species. All of the above stages were observed during each study day. Thin-walled oocysts, microgamonts, and microgametes were seen less frequently than other sexual stages. Microgamonts, macrogamonts, and oocysts attached to the epithelium were all contained in a host cell membrane or within a parasitophorous vacuole. Thin-walled oocysts of C. baileyi were observed for the first time on an ultrastructural level in the respiratory tract of chickens.     

Cryptosporidium canis n. sp. from domestic dogs.

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.     

Coccidia (Eimeria tachyglossi n. sp., E. echidnae n. sp., and Octosporella hystrix n. sp.) in the Echidna, Tachyglossus aculeatus (Monotremata: Tachyglossidae)

Oocysts of Octosporella hystrix n. sp., Eimeria tachyglossi n. sp., and E. echidnae n. sp. are described from the feces of the echidna Tachyglossus aculeatus (Monotremata: Tachyglossidae) from Australia. Eimeria tachyglossi has subspherical oocysts, 26.4 × 23.7 μm in size, with a single oocyst wall; no micropyle; four ellipsoidal sporocysts 13.2 × 9.7, slightly pointed at one end, each containing two sporozoites. Eimeria echidnae has subspherical oocysts, 19.4 × 17.8 in size, with a single oocyst wall; no micropyle; four ellipsoidal sporocysts 9.8 × 7.8, blunt at both ends, each containing two sporozoites. Octosporella hystrix has ovoid or subspherical oocysts 32.9 × 29.7 in size with a thick outer and thin inner oocyst wall; no micropyle; eight sporocysts spherical or slightly subspherical 11.3 × 11.2 each containing two sporozoites lying in embrace, with an extensive granular sporocyst residuum about the equator of the sporocyst. Endogenous stages considered to be of E. tachyglossi at least, were recognized in the lamina propria and epithelium on villi in the small intestine of three echidnas.     

Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens

The structure and infectivity of the oocysts of a new species of Cryptosporidium from the feces of humans are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts. and measure 4.4-5.4 microm (mean = 4.86) x 4.4-5.9 microm (mean = 5.2 microm) with a length to width ratio 1.0-1.09 (mean 1.07) (n = 100). Oocysts were not infectious for ARC Swiss mice, nude mice. Wistar rat pups, puppies, kittens or calves, but were infectious to neonatal gnotobiotic pigs. Pathogenicity studies in the gnotobiotic pig model revealed significant differences in parasite-associated lesion distribution (P = 0.005 to P = 0.02) and intensity of infection (P = 0.04) between C. parvum and this newly described species from humans. In vitro cultivation studies have also revealed growth differences between the two species. Multi-locus analysis of numerous unlinked loci, including a preliminary sequence scan of the entire genome demonstrated this species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further support for its species status. Based on biological and molecular data, this Cryptosporidium infecting the intestine of humans is proposed to be a new species Cryptosporidium hominis n. sp.     

Effect of hyperimmune bovine colostrum raised against Cryptosporidium parvum on infection of guinea pigs by Cryptosporidium wrairi.

Oocysts shedding was markedly reduced in guinea pigs inoculated intraintestinally with Cryptosporidium wrairi sporozoites that had been incubated with hyperimmune bovine colostrum raised to C. parvum when compared with shedding in guinea pigs inoculated with sporozoites incubated in either non-immune bovine colostrum or buffered saline. However oocyst shedding was apparently not reduced in guinea pigs inoculated by gavage with oocysts of C. wrairi and subsequently treated twice daily per os with hyperimmune bovine colostrum. Similarly, oocyst shedding was apparently not reduced by oral treatment with hyperimmune bovine colostrum when treatment was begun simultaneously with inoculation of C. wrairi oocysts.     

Goussia girellae N. Sp. (Apicomplexa: Eimeriorina) in the Opaleye,Girella nigricans1

ABSTRACT Goussia girellae n. sp. is described from the opaleye fish, Girella nigricans. Merogonic stages were observed in the apices of intestinal epithelial cells, in the lamina propria, and in extra-intestinal sites including liver, gills, and spleen. Gamonts were observed in the intestinal epithelial cells. Only unsporulated oocysts were detected in the intestine, and sporulation occurred when feces containing oocysts were incubated for 48 h in seawater at 21°C. Oocysts are elongated (24.8 × 14.7 μm) with a wall about 200 nm thick and have no residuum, micropyle, or polar granule. Sporocysts are ellipsoid (8.5 × 4.5 μm), have a thin two-layered wall approximately 30 nm thick, and consist of two valves joined by a suture. Although moribund opaleye were also infected with Gyrodactylus sp., Cryptobia sp., Cardicola sp., and epitheliocystis organisms (chlamydia), all fish were heavily infected with G. girellae and morbidity was thus attributed to the coccidium.     

Cryptosporidium bovis n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus)

A new species of Cryptosporidium, C. bovis, is described. Oocysts of C. bovis, previously identified as Cryptosporidium genotype Bovine B (GenBank AY120911), are morphologically indistinguishable from those of C. parvum. They are excreted fully sporulated and contain 4 sporozoites, but lack sporocysts. Oocysts measure 4.76-5.35 microm (mean = 4.89 microm) x 4.17-4.76 microm (mean = 4.63 microm), with a length-to-width ratio of 1.06 (n = 50). Oocysts were not infectious for neonatal BALB/ c mice, but were infectious for 2 calves that were previously infected with C. parvum. Oocysts were not infectious for 2 experimentally exposed lambs less than 1 wk of age and were not detected in 42 lambs 2-3 mo of age, but were detected in a 2-wk-old lamb. In an earlier study, 79 of 840 calves on 14 dairy farms in 7 states were found infected with the new species. Most calves were 2-7 mo of age and none exhibited signs of diarrhea. This new species has been found in 10 of 162 calves aged 9 to 11 mo on a beef farm in Maryland. Fragments of the 18S rDNA, HSP-70, and actin genes were amplified by PCR, and purified PCR products were sequenced. Multilocus analysis of the 3 unlinked loci demonstrated the new species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further evidence of species status. Based on these biological and molecular data, we consider this highly prevalent Cryptosporidium that infects primarily postweaned calves to be a new species and propose the name Cryptosporidium bovis n. sp. for this parasite.     

Viability of preserved Cryptosporidium baileyi oocysts

The present study was undertaken to determine the viability and infectivity of oocysts of Cryptosporidium baileyi that had been stored from 1 to 40 months at 4 degrees C preserved in 2.5% potassium dichromate solution. Oocysts of C. baileyi were purified from the feces of experimentally infected chickens using discontinuous sucrose gradients. Subsequently, the purified oocysts were suspended in 2.5% potassium dichromate solution at a concentration of 1 x 10(7) organism/ml, and their viabilities were assessed by nucleic acid staining, histologic examination, and infectivity to 2-day-old chickens. All chickens inoculated with oocysts that had been stored for 1-18 months developed patent infections, while chickens infected with older oocysts remained uninfected. Between 5.8% and 82.2% of the oocysts, stored at 4 degrees C in 2.5% potassium dichromate solution, were found to be viable, as determined by nucleic acid staining. Parasite colonization in the bursa of Fabricius was detected in the microvillus border of bursal epithelium. The finding that C. baileyi oocysts remain infective to chickens for at least 18 months offers important time-saving advantages to investigators who frequently require large numbers of oocysts.     

Induction of folds or sutures on the walls of Cryptosporidium parvum oocysts and their importance as a diagnostic feature.

The proportion of oocysts of Cryptosporidium parvum showing a fold on oocyst walls when incubated with either fluorescent monoclonal antibody or a surface-reactive fluorescent dye was increased by incubating suspensions of oocysts with dimethyl sulfoxide, sucrose, or Hanks' balanced salt solution. Further incubation of sucrose-incubated oocysts with water showed this to be a reversible phenomenon. Oocysts demonstrating this fold after incubation in dimethyl sulfoxide were of the same viability as control oocysts and followed the same excystation dynamics. Despite this fold having been previously described as a suture, we were unable to find any evidence that this pattern of fluorescence highlighted the same suture that has been described in ultrastructural studies. Furthermore, oocysts were observed in which this fold was not always continuous with the gape in the oocyst wall through which the sporozoites had emerged. We propose that this fluorescently highlighted region or fold should no longer be described as a suture and question its validity as a diagnostic feature. When environmental and other samples are being examined for the presence of C. parvum oocysts, objects of appropriate size, shape, and fluorescence which do not demonstrate a surface fold should not necessarily be excluded.     

Early events in the life cycle of the mouse coccidium Eimeria falciformis (Eimer, 1870) Schneider, 1875 in naive and immune hosts

The initial phases of invasion of mammalian coccidia of the genus Eimeria into host tissue are still poorly known. This process, including the passage of oocysts through the intestinal lumen, excystation of sporozoites, their penetration into epithelial cells and migration to the target site was studied in both naive and immune mice infected with Eimeria falciformis. After oral infection, the intact oocysts were transported with enteral contents to the large intestine, where the excystation of sporozoites and their penetration into superficial epithelium took place. The sporozoites subsequently migrated into the epithelium of crypts, which is the specific site of asexual multiplication. The immune status of the hosts did not affect the passage of oocysts, excystation and penetration of sporozoites. However, the migration of sporozoites towards their target site (crypts) was impeded in immune mice and sporozoites tended to remain in superficial mucosa rather than migrate to the crypts.     

A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa).

Cryptosporidium sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 x 4.6 (4.8-5.6 x 4.0-5.0) microns and had a shape index (length/width) of 1.17 (1.04-1.33). Oocysts of C. parvum were similar and measured 5.2 x 4.6 (4.8-5.6 x 4.2-4.8) microns with a shape index of 1.16 (1.04-1.33). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands greater than 100 kDa.